DNA fragmentation in blue mussel (Mytilus edulis) sperm: aquaculture and fisheries implications

The objective of this study was to assess sperm DNA longevity in blue mussels (Mytilus edulis) using a dynamic assessment of sperm DNA fragmentation (SDF) after sperm activation. Mature blue mussels (n = 57) in Vigo (Galicia, Spain) were obtained, specifically rope farmed blue mussels (n = 38) and wild blue mussels (n = 19). After the sperm collection, a subsample was assessed for SDF (0 h), while the rest of the sample was incubated for 6, 9, 12, 24 and 48 h at 15°C, assessing each time point using the Sperm‐Halomax kit (Halotech DNA, Madrid, Spain). The Kaplan–Meier estimator, log‐rank (Mantel–Cox) test and Mann–Whitney U‐test were used for statistical analyses (spss v. 16.0), α = 0.05. The rate of SDF (r‐SDF) between rope farmed and wild blue mussels over 0–6 h incubation was not significantly different (P = 0.278), but was for 6–24 h (P = 0.004). Differences in r‐SDF were observed when comparing the means between the two groups (P < 0.0001). Individual differences in r‐SDF existed among the rope farmed (P < 0.0001) and wild blue mussels (P < 0.0001). Wild blue mussels presented a higher DNA longevity than the farmed blue mussels. Selection of blue mussel males with a low level of sperm DNA damage and greater sperm DNA longevity may result in better fertilization and seed production.     

A molecular tool for parentage analysis in the Mediterranean mussel (Mytilus galloprovincialis)

Estimation of selection response and genealogical tracing in family mixtures require an appropriate tool for parentage analysis. In this study, we tested 19 marker loci for parentage analysis allocation in Mediterranean mussel (Mytilus galloprovincialis). To this aim, we reared families in tanks isolated from wild mussel seed, analysed them using the 19 marker loci and characterized their performances based on Mendelian rules. Probabilities of exclusion of a false parent were estimated for different groups of loci and contrasted to the real paternity assignment. Based on this, we chose nine microsatellites with the highest exclusion probabilities and a real paternity assignment of 99.6%. Next, we analysed 600 individuals reared as in the usual production process, where contamination from wild seed is likely. We obtained a real assignment of 94.7% and were able to identify individuals from the wild as the most likely hypothesis to explain the observed incompatibilities with candidate parents. This information was used to evaluate parental contribution in offspring obtained from gamete mixtures of several parents, which bestowed results of interest for future breeding programs of Mediterranean mussel.     

Effect of different ions on larval metamorphosis of the mussel Mytilus galloprovincialis

Metamorphic responses of pediveliger larvae of Mytilus galloprovincialis to different ions were investigated through a series of bioassays. Effects of tetraethylammonium chloride (TEA) on inductive effects of these above ions were also investigated. Excess ions including Li⁺, Cs⁺, Rb⁺ and Ca²⁺ induced larval metamorphosis at 10^?3 M to 5 × 10^?2 M in 24‐h exposure assays. In continuous exposure assays, only excess Ca²⁺ showed inductive activity and induced 25% metamorphosis at 5 × 10^?2 M. Larval responses to Li⁺ and Rb⁺were inhibited by TEA, while induction of metamorphosis by Cs⁺and Ca²⁺ was independent of the presence of TEA. Thus, these ions used can be useful inducers of larval metamorphosis for application in the aquaculture industry. The finding provides new insights on the biochemical mechanism controlling larval metamorphosis in this species.     

Effect of temperature on survival,growth and development of Mytilus galloprovincialis larvae

Mussel aquaculture is widely prevalent worldwide, but generally relies on natural seed collection, which does not always meet the needs of the producers. Thus, development of mussel hatcheries is of economic interest in some parts of the world, such as Europe; it provides opportunities not only on annual reliability of seed but also on genetic improvements. To broaden knowledge on mussel larval physiology, we carried out temperature treatments (17, 20 and 24 °C) on Mytilus galloprovincialis larvae under laboratory conditions. The trials ended when 30% of the larval population was in the post‐larval stage. The temperature coefficient Q₁₀ indicated a strong relationship between temperature and increase in growth from 17 to 20 °C, but not between 20 and 24 °C. Exposure of M. galloprovincialis larvae to 17 °C resulted in poor growth, low survival and a delayed development and was considered to be inadequate for M. galloprovincialis larval culture. Rearing the larvae at 20 or 24 °C produced better growth, higher survival rates and faster metamorphosis as compared with 17 °C. The temperature region within 20 and 24 °C was suggested as adequate for the mussel M. galloprovincialis larval culture, and implications of these results on the development of commercial hatcheries were discussed.     

Growth variations within a farm of mussel (Mytilus galloprovincialis) held near fish cages: importance for the implementation of integrated aquaculture

Fish farming releases extensive amounts of particulate organic waste that can be exploited by bivalves in integrated culture. We tested if mussels Mytilus galloprovincialis cultured at two depths (1 and 6 m) in a raft, moored 170 m from a fish farm had greater growth than bivalves held 550 m from the fish cages. Mussel growth was monitored monthly, covering the second phase of the culture, from thinning‐out to harvest (March to November 2011). We also studied if fish solid and dissolved nutrients increased the organic content of the seston and chlorophyll‐a levels near the fish cages through weekly samples. Results showed no differences in seston, chlorophyll and physiochemical characteristics of the water among rafts. Maximum growth and Condition Index (CI) occurred during spring–summer (April–August), when mussels had access to greater food quality and quantity. Mussels cultivated close to the cages showed similar shell length, weight and CI compared with mussels distant from the fish farm. Average shell length, meat dry weight and CI at harvest were 76.31 mm, 2.51 g and 23%. Bivalves cultured distant from the fish cages displayed 26% higher biomass than the other raft at the end of the experiment. Differences in biomass were explained by the significantly higher recruitment of mussel seed observed at the raft distant from the fish cages from June to November. The lack of a significant enhancement in growth of the bivalves cultured next to finfish is discussed.     

Effects of larval cryopreservation on subsequent development of the blue mussels,Mytilus galloprovincialis Lamarck

In this study, the effects of three commonly used chemicals, dimethyl sulphoxide (DMSO), ethylene glycol (EG), propylene glycol (PG) and their combinations with trehalose, were evaluated on the cryopreservation of D‐larvae of the blue mussel Mytilus galloprovincialis. The larvae were harvested 30 h post‐fertilization at 21 °C and cryopreserved using a standard protocol in 5%, 10% or 15% of DSMO, EG and PG either as single chemical solutions or in combination with 0.2 M trehalose. Among these cryoprotectants, 5% DMSO resulted in the highest post‐thaw survival rate of 55.3±7.8%, although it did not significantly differ from those with 10% and 15% EG. The addition of 0.2 M trehalose did not improve the post‐thaw larval survival rates in all the combinations assessed. The cryo‐effects on subsequent development were evaluated using the D‐larvae frozen with 5% DMSO. The results showed that cryopreservation affected both larval survival and growth in this species. The relative daily mortality rate was significantly higher in treated than control groups over the period from 3 h post‐thaw to day 11 post‐fertilization. On day 6 post‐fertilization, the average larval length in the treated group was significantly smaller than that in the control. From day 11 post‐fertilization, and onwards, differences in these two traits were not significant between treated and control groups. On day 21 post‐fertilization, about 80% of the larvae in both treated and control groups developed eyes and the normalized survival rate in the treated group was 12.5%.     

Characterization of single‐nucleotide polymorphism markers in the Mediterranean mussel,Mytilus galloprovincialis

The Mediterranean mussel, Mytilus galloprovincialis, is one of the most important aquaculture species in Europe. Appropriate molecular markers are required to evaluate genetic resources and to trace genealogies in breeding programmes for improving mussel culture. Microsatellites have been commonly applied to this purpose in other species. However, Mediterranean mussel microsatellites have demonstrated high frequencies of null alleles that hamper accurate estimates of population parameters and confident parentage inferences. As alternative markers, we have characterized in silico 25 potential single‐nucleotide polymorphism (SNP) markers in the Mediterranean mussel from expressed sequence tag (EST) public databases. The genotyping of SNPs was performed using a single‐base extension approach. Their polymorphism was evaluated in 47 individuals from an Atlantic population. Out of the 25 potential SNPs tested, 12 were technically feasible (producing a single amplicon) and polymorphic. All were biallelic and had an unbiased heterozygosity ranging from 0.160 to 0.504. One SNP was from a mitochondrial gene. The combined potential of nuclear SNPs for parentage assignment gave an exclusion probability of a false couple of parents of 0.9471. These markers will be useful for evaluating resources and tracing genealogies in genetic breeding programmes implemented to solve the main problems of mussel culture.     

Greenlip abalone (Haliotis laevigata Donovan, 1808) sperm cryopreservation using a programmable freezing technique and testing the addition of amino acid and vitamin

This study investigated factors key to the development of sperm cryopreservation in the greenlip abalone Haliotis laevigata using a programmable freezing technique, including (1) permeable cryoprotectant agent (CPA) selection; (2) cooling rate; (3) endpoint temperature; (4) thawing temperature; (5) sperm to egg ratio and (6) sugar, vitamin and amino acid supplementation, using sperm motility, fertilization rate, plasma membrane integrity, mitochondrial membrane potential or acrosome integrity as quality assessment indicators. Results showed that among the permeable CPAs evaluated, 10% dimethyl sulfoxide was the most suitable for greenlip abalone sperm cryopreservation. The highest post‐thaw sperm motility was achieved with the sperm being frozen at a cooling rate of ?5°C min^?1 to ?30°C from 0°C and thawed and recovered in 40°C and 18°C seawater baths respectively. The addition of sugars in 10% dimethyl sulfoxide did not significantly improve the post‐thaw sperm motility and fertilization rate. The addition of 0.6% glycine, 0.2% taurine or 0.02% L‐ascorbic acid, on the other hand, significantly improved the post‐thaw sperm motility. However, only the addition of 0.6% glycine improved the post‐thaw sperm fertilization rate, which was further confirmed by the improvement of the post‐thaw sperm mitochondrial membrane potential and acrosome integrity through flow cytometry analysis.     

Effects of environmental factors and food availability in Northern Aegean sea on the cultivation of Mediterranean Mussels (Mytilus galloprovincialis)

This study used two different collectors made of polypropylene (PP) rope and polyethylene (PE) fishing net to determine Mediterranean mussel's settlement, growth, condition index (CI), meat yield (MY) and shell thickness index (STI) during the period spent between June 2017 and November 2018. With this regard, chlorophyll a, water temperature, salinity, pH and particulate matter were defined through water samples that were collected on monthly basis triplicate. The mean chlorophyll a level was recorded as 0.32 ± 0.31 μg/L, while water temperature was 19.73 ± 5.14°C, salinity was 35 ± 2.070‰, pH was 8.12 ± 0.04, and TPM was 14.91 ± 10.48 mg/L. As a result of the cultivation period of 8 months spent following the first intense grip, the length of the mussels is measured as 31.79 ± 6.20 mm. Based on the Pearson correlation analysis results, the most important environmental parameter affecting Mediterranean mussels growth in length on PP rope collectors is the temperature (p < .05). The STI and CI were determined to be related with environmental temperature parameters, while no determinations could be obtained concerning the correlation of MY with environmental parameters. Assessment of Mediterranean mussels’ growth rate in PP rope collectors proved significant differences (p < .05) between March and November 2018. As no Mediterranean mussels are observed on PE fishing nets during the samplings performed throughout the study period, it is determined that PE fishing nets are not suitable collector types for Mediterranean mussel cultivation.     

Cryopreservation of sperm in farmed blacklip abalone (Haliotis rubra Leach, 1814)

This study developed a technique of sperm cryopreservation using liquid nitrogen (LN) vapour in farmed blacklip abalone Haliotis rubra through evaluating the following five key factors: (1) cryoprotectant agent (CPA) toxicity; (2) cooling temperature; (3) thawing temperature; (4) sperm to egg ratio and (5) sugar addition, using sperm motility or fertilization rate as quality assessment indicators. The results demonstrated that 6% dimethyl sulfoxide (DMSO) was the best single CPA for sperm cryopreservation in this species. The highest post‐thaw sperm motility was achieved when sperm were exposed to LN vapour for 10 min at 5.2 cm above the LN surface and thawed at 60°C and recovered at 16°C in seawater baths. Post‐thaw sperm motility was found to be significantly higher when 6% DMSO was used in combination with 1% or 2% glucose than 6% DMSO alone. Further evaluation of fertilization rate between these CPAs showed that 6% DMSO+2% glucose achieved the highest fertilization rate of 70% at a sperm to egg ratio of 10 000:1.     

Simulation modelling of high‐throughput cryopreservation of aquatic germplasm: a case study of blue catfish sperm processing

Emerging commercial‐level technology for aquatic sperm cryopreservation has not been modelled by computer simulation. Commercially available software (ARENA, Rockwell Automation, Inc. Milwaukee, WI) was applied to simulate high‐throughput sperm cryopreservation of blue catfish (Ictalurus furcatus) based on existing processing capabilities. The goal was to develop a simulation model suitable for production planning and decision making. The objectives were to: (1) predict the maximum output for 8‐h workday; (2) analyse the bottlenecks within the process, and (3) estimate operational costs when run for daily maximum output. High‐throughput cryopreservation was divided into six major steps modelled with time, resources, and logic structures. The modelled production line processed 18 fish and produced 1164 ± 33 (mean ± SD) 0.5‐mL straws containing one billion cryopreserved sperm. Two such production lines could support all hybrid catfish production in the United States and 15 such lines could support the entire channel catfish industry if it were to adopt artificial spawning techniques. Evaluations were made to improve efficiency, such as increasing scale, optimizing resources, and eliminating underutilized equipment. This model can serve as a template for other aquatic species and assist decision making in industrial application of aquatic germplasm in aquaculture, stock enhancement, conservation and biomedical model fish.     

The hidden companion of non‐native fishes in north‐east Atlantic waters

A tropicalization phenomenon of ichthyofauna has been described in the last decades in Galicia (north‐eastern Atlantic), with increasing reports of tropical and subtropical fishes appearing northward this distribution range. A search for parasites was carried out in the digestive tract of two specimens first captured in Galician waters: the prickly puffer Ephippion guttifer (Tetraodontidae) and the African stripped grunt Parapristipoma octolineatum (Haemulidae). Examination of E. guttifer showed high intensity of nematodes, from three different genera: Cucullanus (Cucullanidae), Hysterothylacium (Raphidascaridae) and Anisakis (Anisakidae), with demonstrated pathogenicity to humans. Molecular identification allowed the identification of Anisakis pegreffii, already described in the area, and first reports for European waters of Cucullanus dodsworthi, Hysterothylacium reliquens and a new Hysterothylacium sp. P. octolineatum showed a far lower level of parasitization, with two Hysterothylacium larvae, genetically identified as Hysterothylacium deardorffoverstreetorum, also its first report in the eastern Atlantic. Thus, possible ecological impact of the occurrence of two non‐native individual fishes in a new area could be remarkably higher if we see this issue through the lens of the parasitological perspective, as far as only two individual fish can harbour more of one hundred nematode parasites belonging to different species, most of them also new species for that area.     

Temperature‐dependent feed requirements in farmed blue mussels (Mytilus edulis L.) estimated from soft tissue growth and oxygen consumption and ammonia‐N excretion

Feed requirements were estimated from specific growth rates in standardized soft tissue dry weight (SGR_DW’) and atomic O:N ratios for mussels fed seven rations of microalgae (5–735 μg C h^?1 ind^?1) at 7 and 14°C respectively. The mean oxygen consumption and ammonia‐N excretion rates were significantly higher at 14°C (0.29 μg O² and 27.3 μg N ind^?1 h¹) compared with those at 7°C (0.16 μg O² and 11.4 μg N ind^?1 h^?1) (P < 0.05), resulting in O:N ratios between 3 and 45 at 7°C and 7 and 28 at 14°C. Low O:N ratios indicate protein catabolism and an unfavourable condition, whereas high ratios indicate that carbohydrate is the primary energy source. The measured SGR_DW’ suggests minimum feed requirements of ~240 and ~570 μg C ind^?1 h^?1 for weight maintenance at 7 and 14°C, with corresponding O:N ratios of 24 and 16, respectively, indicating a more stressed condition at 14°C. A 0.5% SGR_DW’ day^?1 was obtained by ~565 (O:N = 29) and ~680 (O:N = 23) μg C ind^?1 h^?1 at 7 and 14°C respectively. A positive and significantly higher SGR_DW’, with the lowest feed ration at 7°C compared with a negative SGR_DW’ at 14°C (P < 0.05), indicated that storage time can also possibly be prolonged at low temperatures if the mussels are not fed.     

Evaluation of growth performance and intestinal barrier function in Arctic Charr (Salvelinus alpinus) fed yeast (Saccharomyces cerevisiae), fungi (Rhizopus oryzae) and blue mussel (Mytilus edulis)

Arctic charr (Salvelinus alpinus) were fed for 99 days on experimental diets with 40% of fish meal replaced, on a crude protein basis, with intact yeast (Saccharomyces cerevisiae) (ISC), extracted yeast (ESC), Rhizopus oryzae fungus (RHO) or de‐shelled blue mussels (Mytilus edulis) (MYE). The fish were evaluated for growth performance, nutrient digestibility and fish intestinal function. Growth performance, retention of crude protein and sum of amino acids were not affected in fish fed diets ISC or MYE compared with those fed the reference (REF) diet. However, fish fed diet ISC displayed decreased digestibility of crude protein and indispensable amino acids and decreased intestinal barrier function compared with fish fed the REF diet. Fish fed diet ESC exhibited decreased growth performance and protein retention, but had comparable digestibility to fish fed the REF diet. Fish fed diets MYE and RHO showed similar performance in terms of growth, nutrient digestibility and intestinal barrier function. Overall, the results indicated that blue mussel and intact S. cerevisiae yeast are promising protein sources for Arctic charr.     

Embryonic and larval development of a hybrid between kelp grouper Epinephelus moara ♀ × giant grouper E. lanceolatus ♂ using cryopreserved sperm

Hybridization was used to take advantage of desirable traits in offspring. In the present study, we applied the cryopreserved Epinephelus lanceolatus sperm into interspecific hybridization with E. moara. Successful hybridization between these two species was achieved and cultured in 23–24°C seawater (34‰). There was no difference in survival rate between hybrid (E. moara ♀ × E. lanceolatus ♂) and non‐hybrid (E. moara ♀ × E. moara ♂) at 24 hrs post hatch (HPH), but less hybrid (14.35 ± 8.02%) hatched than non‐hybrid (93.60 ± 1.65%), which might be due to irregularity cell cleavage and skeletal deformities from the formation of embryonic body to the later embryonic development. Similar phenomenon was found in hybrid embryos from fertilization with fresh sperm, indicating that species variations between parents, rather than cryopreserved sperm, resulted in deformities in embryos. Mean hatch time of the hybrid was 1 hr faster than that of E. moara. The hybrid was 1.95 ± 0.06 mm in total length when newly hatched and reached 38.00 mm at 58 days post hatch (DPH), which showed faster growth than E. moara (recorded in the previous study). Considering its faster growth, E. moara × E. lanceolatus hybrid was a potential breeding production in aquaculture. Over 212,000,000 larvae have been produced and launched in the market since 2015. The results of this study also shed some lights on further comparative studies in grouper hybrid performance.     

Development of a two‐step,non‐probed multiplex real‐time PCR for surveilling Vibrio anguillarum in seawater

Vibrio anguillarum is an aggressive and halophilic bacterial pathogen most commonly originating from seawater. Vibrio anguillarum presence in fisheries and aquaculture facilities causes significant morbidity and mortality among aquaculture species primarily from haemorrhaging of the body and skin of the infected fish that eventually leads to death, collectively recognized as the disease vibriosis. This study served to develop a non‐probe, multiplex real‐time PCR assay to rapidly detect V. anguillarum presence in seawater. Specific primers targeting genes vah1, empA and rpoN of V. anguillarum were selected for multiplex reaction among 11 different primer sets and the extension step was eliminated. Primer concentration, denaturation time as well as annealing time and temperature of DNA amplification were optimized, thus reducing reaction duration. The two‐step, non‐probed multiplex real‐time PCR set forth by this study detects as little as 3 CFU mL^?1 of V. anguillarum presence in sea water, without enrichment cultivation, in 70 min with molecular precision and includes melting curve confirmation.     

Immune responses of Litopenaeus vannamei to non‐ionic ammonia stress: a comparative study on shrimps in freshwater and seawater conditions

To investigate the effect of non‐ionic ammonia (NH₃‐N) stress (0.1 and 0.5 mg L^?1) on the immunity of Litopenaeus vannamei cultured in long‐term freshwater, the total haemocyte count (THC), the activity of phenoloxidase (PO), nitric oxide synthase (NOS), superoxidase dismutase (SOD) and the content of malondialdehyde (MDA) were determined and further compared with those of seawater shrimps. The results showed that NH₃‐N stress significantly reduced THC and the activity of PO and SOD (P < 0.05). Under 0.1 mg L^?1 NH₃‐N stress, NOS activity increased first and then decreased significantly, while it dropped dramatically under 0.5 mg L^?1 NH₃‐N stress (P < 0.05). During NH₃‐N stress, MDA content increased continuously, and the MDA content in hepatopancreas of freshwater shrimps was higher than that of seawater shrimps. It was concluded that NH₃‐N stress significantly influenced the non‐specific immunity and could also upset the balance of antioxidant system of L. vannamei in both freshwater and seawater shrimps. Compared with in seawater, the shrimps in freshwater were more vulnerable to NH₃‐N stress because of higher lipid peroxidation and lower immunity.     

An extender solution for the short‐term storage of Litopenaeus vannamei sperm to be used in artificial insemination

Some shrimp hatcheries use artificial insemination (AI) to improve the male to female ratio in their breeding populations. We describe a sperm extender solution, which allows the short‐term storage of diluted sperm in Litopenaeus vannamei, and its use in an artificial insemination process. We also evaluate its fertilization capacity. An AI experiment was designed using two, one, or half spermatophore segments. We tested four treatments involving three different male:female ratios: Natural mating (1:1), Regular and Regular diluted (1:2) and Half diluted (1:4). Data analysis revealed that the number of nauplii produced per mating was affected by treatment, with Regular (158 420) performing better than Half diluted (112 864) (P < 0.05), but with no differences between the latter and Regular diluted (130 340) (P > 0.05). A binomial variable named female success (FS) was defined as successful when the number of nauplii obtained per mate was ≥25 000. Analysis showed differences for FS across treatments (P < 0.001), but not between Regular (79.2%), the hatchery conventional AI technique and Half diluted (60.4%), maybe due to sample size. Since the number of nauplii per mate is crucial to consider AI successful, it is necessary to improve this AI technique before it can be used in the shrimp industry.     

Characterization of phenotype variations of luminescent and non‐luminescent variants of Vibrio harveyi wild type and quorum sensing mutants

Vibrio harveyi, a luminescent Gram‐negative motile marine bacterium, is an important pathogen responsible for causing severe diseases in shrimp, finfish and molluscs leading to severe economic losses. Non‐luminescent V. harveyi obtained by culturing luminescent strains under static and dark condition were reported to alter the levels of virulence factors and metalloprotease gene and luxR expression when compared to their luminescent variants. Presently, we conducted an in vitro study aiming at the characterization of virulence‐related phenotypic traits of the wild‐type V. harveyi BB120 strain and its isogenic quorum sensing mutants before and after switching to the non‐luminescent status. We measured the production of caseinase, haemolysin and elastase and examined swimming motility and biofilm formation. Our results showed that switching from the bioluminescent to the non‐luminescent state changed the phenotypic physiology or behaviour of V. harveyi resulting in alterations in caseinase and haemolytic activities, swimming motility and biofilm formation. The switching capacity was to a large extent independent from the quorum sensing status, in that quorum sensing mutants were equally capable of making the phenotypic switch.     

Production of inbred larvae through self‐fertilization using oocytes and cryopreserved sperm from the same individuals after sex reversal in eastern oyster Crassostrea virginica

The eastern oyster Crassostrea virginica can change sex which makes self‐fertilization possible if sperm can be cryopreserved. In this study, small (~1 year old) and large (~2–3 years old) oysters were biopsied for sperm collection. Survival of the biopsied oysters after 1 year was 50% for small oysters and 17% for large oysters. Oocytes were collected from sex‐reversed females, and self‐fertilized with cryopreserved sperm. Of the 24 cryopreserved samples, 14 individuals had ≤1% fertility when crossed with oocytes from unrelated females, indicating that the cryopreserved sperm had reduced fertility. The other 10 individuals had a fertility of 39 ± 25% when crossed with oocytes from unrelated females (non‐selfing), but showed a significantly lower success of self‐fertilization (12 ± 16%) (P = 0.008), while aliquots of the same oocytes had a fertilization of 83 ± 11% when crossing with fresh sperm. Larvae were produced at day 3 in the self‐fertilized families (12–94% of the fertilized oocytes), and survived to eyed‐larvae stage at days 11–14. Genotyping with 9 microsatellite markers confirmed that the larvae resulted from self‐fertilization in four families. This study demonstrated the feasibility of creating self‐fertilized inbred lines of oysters by use of non‐lethal sperm collection and cryopreservation.